Brain Aβ42 Targeted Dual-functional Nanoparticles For The Treatment Of Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders and the main causes of death in developed countries. Currently, therapeutic drugs such as cholinesterase inhibitors or glutamate receptor antagonists can only meliorate the symptoms instead of curing the disease, and an actual therapeutic strategy for AD is still lacking. Although numerous biotech drugs have been developed from studies of the molecular pathogeneses of AD, such as nerve growth factor (NGF) and P-sheet breaker peptides, few of them can be used in clinical treatment. There are two main obstacles exists for their use:firstly, the blood-brain barrier (BBB) prevent most exogenous substances entering the brain; secondly, the unselected distribution of drugs after transporting into the brain may result in severe side effects on normal brain. Therefore, a precise delivery of drugs to the AD lesions, to increase the drug efficiency and thus reduce the side effects, is one of the focuses in AD therapy.Considering these problems, "dual targeting" strategy is applied in this project for AD therapy. Dual functional ligands are modified to the surface of nano-delivery system. The first ligand should be chosen to enhance BBB penetration of the delivery system, while the second ligand should have high affinity on disease region of AD and guild the delivery system to the lesion site.The presence of Aβ peptide deposition in senile plaques is one of the identified neuropathological hallmarks of AD, which is widely considered as the most appropriate biomarker and the ideal diagnostic and therapeutic target for AD. Aβ42 is the predominant species of Aβ peptide (approximately 96% the total) and its monomer is the most toxic isoform. Therefore, TGN peptide (consensus peptide sequence "TGNYKALHPHNG") and QSH peptide (consensus peptide sequence "QSHYRHISPAQV") were chosen as the two ligands specifically targeting BBB and senile plaques formed by Aβ42 deposition, respectively. These two peptides were modified to the surface of poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) nanoparticles to develop a dual-functional delivery system for the precise drug delivery to the brain senile plaques. The in vitro, ex vivo and in vivo experiments were conducted for the conformation of the two-stage targeting effects of the delivery system. Afterwards, a β-sheet breaker H102 (consensus peptide sequence "HKQLPFFEED") was encapsulated into the nanoparticles to see whether this dual-functional delivery system could achieve better AD therapeutic effects compared with non-modified or single-modified nanoparticles.In the first part, the PEG-PLA nanoparticles (NP) were prepared using the emulsion/solvent evaporation method. A maleimide-thiol coupling reaction was conducted for the conjugation of TGN and QSH to the surface of nanoparticles to obtain the dual-functional delivery system (TQNP). The results of X-ray photoelectron spectroscopy (XPS) experiments proved the success conjugation of the two peptides. Bend.3 cellular uptake and mice in vivo imaging were performed for the density optimization of TGN, while Thioflavin T (Th-T) binding assay, surface plasmon resonance (SPR) experiments were conducted to evaluate the best modified density of QSH. The satisfactory targeting effects were achieved when the molar ratio of the maleimide on the nanoparticles and the thiol on peptide being 3 for both TGN and QSH (T3Q3NP).In the second chapter of first part, the dual-targeting effect was confirmed by brain distribution studies of nanoparticles and ex vivo imaging, while the mechanism of cellular uptake and cytotoxicity of the delivery system were also evaluated. T3Q3NP could obviously inhibit the formation of Aβ42 fibrils when incubated with Aβ42 monomers, and coumarin-6-loaded T3Q3NP could achieve excellent co-location with brain amyloid plaques of AD model mice, which indicating that QSH modified nanoparticles had great affinity with Aβ42. After administered with DiR-loaded NP, T3-NP and T3Q3-NP, the hippocampus concentration of T3Q3-NP was significantly stronger than that of NP and T3-NP, with AUC value being 3.43 and 1.61 times of that in NP and T3-NP, respectively. These results indicated that T3Q3-NP was transported into the brain and realized a good amyloid plaques targeting effect. The results of cellular localization and the uptake mechanism showed that lysosomes and caveolaes played important roles in the cellular uptake process, and the uptake of the nanoparticles was energy-dependent. MTT assay was used to evaluate the cytotoxicity of nanoparticles. At every concentration (0.1～10 mg/mL), the viability of both bEnd.3 and PC12 cells was above 90% for all studied nanoparticles, indicating they had uniformly low cytotoxicity. Therefore, the PEG-PLA nanoparticles modified with TGN and QSH might be promising drug carriers with good safety.In the second part, H102 peptide was encapsulated into the nanoparticles. The nanoparticles were modified with TGN or QSH to obtain TNP/H102, QNP/H102 and TQNP/H102. The mean particle sizes of the H102-loaded nanoparticles were around 120 nm, with zeta potential being about -28 mV. The loading capacities of these four H102-loaded nanoparticles were similar, being around 0.54-0.61% with the encapsulation efficiency of about 57.68-65.54%. Encapsulation of H102 into nanoparticles could obvious increase the stability of H102 in both plasma and brain homogenates. The in vitro release of nanoparticles was investigated in both PBS and plasma. During one day incubation, nearly 65% of H102 was released in plasma, while less than 40% of H102 was released in PBS, presenting the effect of sustained release.In the second chapter of second part, the amount of H102 in the brain tissues and blood was determined with HPLC-Mass spectrometry. The three H102-loaded nanoparticles (NP/H102, TNP/H102 and TQNP/H102) possessed similar concentration-time profiles in the blood. The cerebrum and cerebellum maximum concentrations of H102 in T-NP and TQ-NP were about 1.8 times higher than the concentration in NP; the AUC values were 1.33-1.49 times higher, suggesting that more H102 was accumulated in the brain tissues after encapsulated into the TGN modified nanoparticles. The drug targeting index (DTI) of TQ-NP/H102 in the hippocampus of AD model mice (3.67) was significantly higher than that of T-NP/H102 (1.94), presenting the excellent hippocampus-targeting effect of TQ-NP for H102 delivery.In the third chapter, the pharmacodynamics of H102 formulations was studied. AD model mice were built by bilateral injection of Aβ42 into the hippocampus, and Morris Water Maze was used to evaluate the memory improvement and neuroprotective effects of H102 formulations on AD model mice. When treated with T-NP/H102 and TQ-NP/H102, especially for the high-dose groups of TQ-NP/H102 (250), mice showed obviously increased learning ability. The results in the measurement of biochemical indexes and the observation of histology showed that T-NP/H102 and TQ-NP/H102 could exert obvious protective effects on hippocampus neurons in a dose-dependent manner. TQ-NP/H102, both high-dose and medium-dose, could achieve excellent prevention of Aβ-induced neuronal death and even help renew the neurons to the healthy state. Moreover, the therapeutic effects in the groups of T-NP/H102 (250) and TQ-NP/H102 (100) were alike, which revealed the better AD amelioration of TQ-NP/H102 formulation.In the last chapter, the short-term toxicity of TQNP/H102 was investigated following repeated intravenous administration for three weeks. The indexes of hemotology in mice were found normal, and no obvious histopathological changes were observed in the main organs (heart, liver, spleen, lung and kidney) of mice. These results indicated that TQNP/H102 could be a safe drut delivery system for AD therapy.